Several strategies exist for responding to infection, immune challenges, inflammation, and trauma in a host. One mechanism by which the host attempts to respond to these challenges is through the upregulation of cytokines, nonantibody proteins that act as intercellular regulators. Some cytokines, known as proinflammatory cytokines, counteract the challenges to the host by enhancing the disease in the hopes of ridding the host of the challenge and host cells damaged by the challenge. Proinflammatory cytokines include, but are not limited to, interleukins (IL), such as IL-1, IL-6, IL-8, and IL-18, and tumor necrosis factor (TNF).
When released, the proinflammatory cytokines have the effect at the site of injury of increasing the release of antibodies and their compliments, T and B cell activation, the adhesion of platelets to blood vessel walls, and extravascuarization of lymphocytes and macrophages. These changes lead to a localized environment at the site of injury including fever, tissue injury, tumor necrosis, induction of other cytokines and immunoregulation and apoptosis. This localized response is toxic not only to the source of the challenge to the host but also to the host cells within the penumbra of the proinflammatory cytokine response. Thus, it is not surprising that on a systemic level, such as may occur during overwhelming infection or serious trauma to the host, many of these proinflammatory cytokines are harmful to the host producing fever, inflammation, tissue destruction, and, in some cases, shock and death.
Representative of the action of the various proinflammatory cytokines is TNF. TNF is a proinflammatory cytokine produced by many cell types, including macrophages, monocytes, lymphoid cells and fibroblasts in response to inflammation, infection, and other environmental challenges. TNF elicits a wide spectrum of cellular responses, including fever, shock, tissue injury, tumor necrosis, anorexia, induction of other cytokines and immunoregulatory molecules, cell proliferation, differentiation and apoptosis. When released TNF has an effect at the site of injury of increasing the release of antibodies and their compliments, T and B cell activation, the adhesion of platelets to blood vessel walls, and extravascuarization of lymphocytes and macrophages. Systemically, TNF acts upon the hypothalamus and liver. TNF stimulates the hypothalamus to release corticotropin releasing hormone, suppress appetite and induce fever. In response to TNF, the liver initiates an acute phase response resulting in the synthesis of several proteins including C-reactive protein, coagulation factors and compliment factors. Also, TNF induces insulin resistance. In the defined area of injury or infection, TNF is vital to removing the particular infectious agent and adapting the body's immune response to the particular injury.
On a systemic level, however, in which TNF as well as other proinflammatory cytokines may be present at higher concentrations or for prolonged times, TNF can have deleterious effects on the body. At high concentrations TNF activates an IL-1 & Il-6 cascade that results in cachexia (wasting). Additionally, TNF can lead to systemic edema, hypoproteinemia, and neutropenia which can result in disseminated extravascular coagulation and eventually multiple organ failure. In chronic diseases such as cancer, TNF can also interfere with vital endogenous functions within the host. For example, TNF may interfere with the ability of endogenous erythropoietin to maintain the hematocrit of the host, leading to a condition referred to as the anemia of chronic diseases (ACD). A typical course of treatment with recombinant erythropoietin may not counteract the effects of the proinflammatory cytokine, thereby requiring the administration of elevated doses of recombinant erythropoietin just to maintain the normal hematocrit of the host. Beyond the additional costs associated with the increased dosing, there is also the risk of adverse side effects from the increased doses of erythropoietin such as thrombosis. In addition to the conditions detailed below, proinflammatory cytokines, including, but not limited to, TNF are associated with diseases such as chronic inflammation, bacterial septic shock, bacterial toxic shock, graft vs. host disease, and HIV infection and AIDS.
Sepsis
Sepsis is the body's response to any kind of infection, e.g. bacterial, viral, parasitic, or fungal. Sites of infection are typically the lungs, the urinary tract, the abdomen, and the pelvis. In some cases, however, the actual site of infection cannot be detected. Although sepsis was once thought to be a systemic inflammatory response, it is now recognized that sepsis also includes prothrombotic diathesis and impaired fibrinolysis.
Once sepsis commences, widespread inflammation and clotting occurs throughout the body. Whereas in a healthy body, immune modulators would be released to fight the infection and heal the body, in sepsis, an overabundance of immune, regulators is released. The release of proinflammatory cytokines such as TNF, interleukin-1, and interleukin-18 lead to the inflammation of endothelial linings, elevation of the core temperature, loss of appetite, and anemia. In addition, inflammation of the lining of blood vessels activates the blood clotting process. Because sepsis decreases the body's natural production of protein C, which regulates blood clotting and controls inflammation, the body's ability to break down the formed blood clots is suppressed. This suppression leads to clotting in vital organs, limbs, fingers, and toes, which, in turn, leads to organ failure or gangrene.
Sepsis may present itself in varying degrees. For example, in cases of severe sepsis, which occurs when acute organ dysfunction or failure results, the body's normal defense reaction goes into overdrive, setting off a cascade of events that can lead to widespread inflammation and blood clotting in tiny vessels throughout the body. Septic shock occurs when a patient with severe sepsis experiences cardiovascular system failure. This failure causes the blood pressure to drop, which, in turn, deprives vital organs of an adequate oxygenated blood supply. Septicemia is a sepsis that has an infection in the bloodstream itself. In fact, septicemia may cause ischemia, i.e., poor blood supply to at least one organ. For example, when blood flow to the kidneys is reduced to dangerously low levels for substantial time period, ischemic acute renal failure (ARF) may develop. The depressed blood flow also results in necrosis, or tissue death, in affected organs.
Providing the source of the sepsis can be identified, many cases of sepsis will respond to treatment. Once isolated, a treatment regime specific to the cause of infection is initiated. Known treatment includes the use of antibiotics, surgical excision of infected or necrotic tissues, drugs that increase activated protein, and steroids (in cases of septic shock). For example, a typical course of sepsis treatment includes administration of a broad spectrum antibiotic until the cause of infection is isolated. However, the mortality rate of sepsis patients remains relatively high in cases of sepsis where the cause and/or area of infection is not ascertainable.
Depending on the severity of sepsis, anti-infection agents, draining techniques, fluids, drugs to raise the mean arterial blood pressure (MAP) such as norepinephrine and phenylephrine, drugs to improve renal function such as dopamine, drugs to increase oxygen delivery and oxygen consumption such as dobutamine and epinephrine, mechanical ventilators to support breathing, and dialysis for kidney failure may be used in the course of treatment. In addition, pharmacological agents that have been shown to have beneficial effects on immune responses following shock and sepsis include ATP-MgCl2, nonanticoagulant heparin, calcium channel blockers, chloroquine, cyclooxygenase inhibitors, PAF antagonists, anti-inflammatory cytokines, growth factors, dietary manipulation, anti-TNF antibodies, activated protein C (Xigris®, Eli Lilly, Indianapolis, Ind.), and sex hormones. Recovery from sepsis is greatest when the condition is quickly diagnosed and promptly treated.
Recombinant erythropoietin (rhu-EPO), commercially available under tradenames PROCRlT® (from Ortho Biotech Inc., Raritan, N.J.), EPOGEN® (from Amgen, Inc., Thousand Oaks, Calif.), and NEORECORMON (from Roche, Basel, Switzerland)has also recently been investigated with regard to treatment of various conditions related to sepsis. In addition, U.S. Patent Publication No. 2003/0083251 generally discloses the use of rhu-EPO to aid in the regeneration of renal tubular cells and prevention of apoptosis of the renal tubular cells in order to treat patients with ischemic ARF. Furthermore, US Patent Publication No. 2002/0061849 generally discloses the use of rhu-EPO to aid in the treatment of inflammation in a non-ischemic condition in one or more organs. However, because of erythropoietin's erythropoietic effects—increased hematocrit, vasoconstriction, hyperactivation of platelets, pro-coagulant activity, and increased production of thrombocytes—treatment with rhu-EPO poses additional risks given the widespread clotting in vital organs, limbs, fingers, and toes that is associated with sepsis.
Adhesions.
In addition to sepsis, proinflammatory cytolines, such as TNF, have been associated with the formation of adhesions, abnormal fibrous bands or connections between organs and other structures of the body, as well. Adhesions may be a complication of, or related to, sepsis but also may occur independently. For example, adhesions may form as a result of surgery, trauma, infection, chemotherapy, and radiation. In fact, adhesions are almost an inevitable outcome of surgery, i.e., about 93 percent of patients who have undergone abdominal surgery suffer from adhesions to some degree (compared with adhesion formation in about 10.4 percent of patients who had never undergone a previous abdominal operation). See D. Menzies and H. Ellis, Intestinal Obstruction from Adhesions—How Big is the Problem?, ANN. R. COLL. SURG. ENGL. 72: 60-3 (1990).
The formation of adhesions can cause severe pain and apply unnatural pressure or tension on organs or other structures of a patient. For example, adhesions in the abdominal region of the body may cause the intestines of a patient to become trapped or squeezed between organs or other structures of the body. In some cases, the intestines may become blocked or significantly obstructed due to nearby adhesions.
The formation of these abnormal connections between two parts of a body leads to a host of other conditions. For example, as cesarean sections are becoming a more common method of childbirth, women who undergo this major abdominal surgery are likely to form adhesions and, as a result, experience chronic pelvic pain. In addition, adhesions involving female reproductive organs may lead to infertility and dyspareunia.
A number of agents have been researched in connection with preventing and treating adhesions, e.g., dextran, corticosteroids, phosphatidylcholine, phospholipase inhibitors, non-steroidal anti-inflammatory drugs, proteoglycans, heparin, and tissue plasminogen activator. See, e.g., C. L. Kowalczyk and M. P. Diamond, The Management of Adhesive Disease, in PERITONEAL ADHESIONS 315-324 (K. H. Treutner and V. Schumpelick, eds., 1997). Some, but not all of these agents, are believed to be effective in the treatment of adhesions because of their ability to interfere with coagulation and fibrinolysis. Clinical experience with the majority of these agents, however, is limited due to bleeding complications. In addition, hyaluronic acid derivatives have been shown to prevent postsurgical adhesions, particularly in the intra-abdominal area. See, e.g., J. M. Becker et al., Prevention of Postoperative Abdominal Adhesions by a Sodium Hyaluronate-based Bioresorbable Membrane: A Prospective, Randomized, Double-blind Multicenter Study, in J. AM. COLL. SURG. 183 297-306 (1996). Furthermore, beta-glucan, which is a glucose polymer that binds with high affinity to the receptors on monocytes and neutrophils in a competitive manner, has been shown to have a reducing effect on the frequency of adhesion after experimentally developed intraabdominal sepsis in Wistar rats. A. Bedirli et al., Prevention of Intraperitoneal Adhesion Formation Using Beta-Glucan After Ileocolic Anastomosis in a Rat Bacterial Peritonitis Model, in AM. J. SURG. 185 339-343 (2003).
Surgery may also be used as a course of treatment for adhesions. Generally, a physician will perform surgery to sever the adhesions from the organ or other part of the body. Given that adhesions are often a complication of surgery, however, surgery to remove adhesions frequently results in the formation of new adhesions. While some surgical procedures involve placement of sleeves over organs adjacent to the areas affected by the surgery and thus, help to prevent adhesions involving these organs, such procedures have had mixed results. In addition, the organ sleeves also require additional surgery to remove the sleeves.
Thus, despite the increased awareness with regard to adhesions, research into treatment methods have met with limited success. Many physicians are unwilling or unable to address the treatment of adhesions and many insurance companies are unwilling to pay for treatments that are, at best, marginally successful.
Wound Healing.
Healing is an essential process of the body that reestablishes the integrity of damaged tissue. This process is often viewed in terms of wounds, ulcers or lesions of the skin resulting from various causes such as trauma, surgery, pressure (bed sores), bums, diabetes, etc. The severity of the wounds is characterized by the extent the wound penetrates the skin. Stage I wounds are characterized by redness or discoloration, warmth, and swelling or hardness. Stage II wounds, partial thickness wounds, penetrate the epidermis and superficial dermis of the skin. Stage III wounds, fall thickness wounds, penetrate through the dermis of the skin but do not penetrate the membrane separating the skin from deeper organs. Stage IV wounds involve damage to the underlying muscle or bone.
Although all wounds heal through the same process: inflammation, epithelialization, angiogenesis, and the accumulation of matrix; the ease with which the wound heals is largely based on the severity of the wound and the health of the wounded individual. In general, Stage I and Stage II wounds heal through the regeneration of epithelial cells by the underlying dermis. Whereas, Stage III and IV wounds heal through the production of a scar. Proinflammatory cytokines, such as TNF, play a role in the healing of wounds, however, it is speculated that TNF may have an adverse effect on the accumulation of collagen in the healing wound and ultimately on the time the wound takes to heal and the strength of the repaired tissue.
Several therapeutics as well as therapeutic methods have been developed to assist the body in healing wounds. Several compounds are considered to have a therapeutic effect on wound healing including, but not limited to, growth factors (epidermal growth factor, Insulin-like Growth Factor, human growth hormone, fibroblast growth factor, vascular endothelial growth factor, interleukin-6, and interleukin-10), nutritional supplements (arginine, glutamine, vitamin C, vitamin B5, Bromelain, Curcumin, zinc, copper), and herbal supplements (aloe vera, Centella). Furthermore, various therapeutic methods including, but not limited to, hyperbaric oxygen therapy, whirlpool therapy, ultrasound therapy, electrical stimulation, and magnetic therapy have been utilized to aid the body in healing wounds.
If a wound does not heal properly or fails to heal at all it can lead to several complications chief among them scarring and infection. Depending upon the severity of the wound, the body may generate scar tissue in healing the wound. Aside from the aesthetic concerns of a scar, the scar may impair movement of the individual depending upon its severity. Additionally, a wound presents an opportunity for bacteria and other infectious agents to enter the body. Depending upon the severity of infection it may spread and become systemic leading to sepsis or septicemia.
Rhu-EPO has also been investigated for its possible healing effects in rat models of random ischemic flaps. For example, rhu-EPO has been shown to reduce necrosis, decrease neutrophil infiltration, and prevent increased temperature with regard to ischemic skin flap injuries. See M. Buemi et al., Recombinant Human Erythropoitein Influences Revsacularization and Healing in a Rat Model of Random Ischaemic Flaps, ACTA DERM VENERBOL, 82: 411-417 (2002). This finding suggests that rhu-EPO administration can improve the wound healing process, both in early and late stages of injury, by reducing the inflammatory response, increasing the density of capillaries in ischemic flaps and allowing earlier repair of a damaged area. However, as mentioned above, because rhu-EPO has erthyropoictic activity, the use of rhu-EPO for treatment of these conditions may cause a greater degree of clotting or complications than already initiated by the healing process.
In sum, no one agent or treatment strategy has demonstrated sufficient value for the management of sepsis cases, the incidence of sepsis, the formation of adhesions, wound healing or general inflammatory conditions. In fact, the mortality associated with sepsis and related conditions remains high. Every year, approximately 215,000 people die from severe sepsis and one out of every three patients who develop severe sepsis will die within a month. And, cases of sepsis are expected to rise in the future due to the increased awareness of the condition and sensitivity for the diagnosis, the number of immunocomprorrised patients, the use of invasive procedures, the number of resistant microorganisms, and the growth of the elderly population. In addition, the chronic pain associated with adhesions and general inflammatory conditions is often untreated due to the lack of a successful treatment strategy.
Thus, there exists a need in the art for method and therapeutics for treating, preventing, delaying the onset of, and reducing the effects of proinflammatory cytokines for the purposes of limiting the penumbra of their action and further addressing their systemic effect. In particular, a need exists for treating, preventing, delying the onset of, and reducing the effects of proinflammatory cytokines in conditions of sepsis, adhesions, wounds, chronic disease and general inflammatory conditions. In addition, it would be beneficial to provide methodoligies that have the ability to repair or prevent damage to tissue in ischemic conditions.